Genetic analysis of Leishmania donovani tropism using a naturally attenuated cutaneous strain.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Resumed from a prior worker's checkpoint (align+slrnaseq done, variant/CNV/DE pending). Fixed a Java8(GATK3.8)/Java11+(SnpEff5.1) version conflict by installing an isolated openjdk17 for the SnpEff steps only. Completed full pipeline: GATK3.8 joint HaplotypeCaller -> hard-filter -> SnpEff annotate -> classify_variants.py (Table2/3) and somy_cnv.py (Table1); edgeR Table4 was already done. Table3 (5 specific variant positions) reproduces EXACTLY. Table1 CNV: 2/9 loci within-tolerance (chr16,chr23), 5/9 correct-direction-but-off-magnitude, 1/9 genuine mismatch (chr19, no depletion seen), 1/9 (chr22/A2) is the paper's OWN documented non-reproducible locus (manual curation due to ref misassembly) reported as reference only. Table2 SNP/indel counts: correct category structure but ~40-60% of reported magnitude (likely differing variant-calling stringency vs the paper's unspecified original pipeline). Table4 DE: 4/8 highlighted genes match direction+magnitude, 3/8 no signal in my pooled edgeR design vs paper's per-stage contrasts (documented design mismatch), 1/8 direction only. Not attempted: wet-lab/manual curation results (chr22 A2 locus exact values, any non-pipeline validation experiments) - out of scope per brief.
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- Reproduced
- 2026-08-01
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-08-01no human curator yet
- Last updated
- 2026-08-01
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusWhy do most Leishmania species cause cutaneous infection while others visceralize? The study tests whether closely related L. donovani (MON-37) clinical isolates from Sri Lankan cutaneous (CL-SL) and visceral (VL-SL) leishmaniasis patients differ genetically in ways that determine visceral organ tropism.
- ★ The CL-SL L. donovani isolate is severely attenuated for survival in visceral organs (liver, spleen) of BALB/c mice compared with the VL-SL isolate, while inducing transient footpad swelling that VL-SL does not. finding
- ★ Gene deletions and CL-SL-specific pseudogenes are not responsible for the difference in disease tropism; no gene deletions were detected in either genome and only 5 genes are unequally affected by frame shift/stop-site change, with no informative homozygous CL-SL-specific pseudogene. finding
- ★ SNPs and/or gene copy number variations play a major role in the altered pathology/tropism of the CL-SL strain. mechanism
- ★ Expression of the VL-SL Rag C gene (LdBPK_366140.1/366149, ras-like small GTPase in the mTOR pathway) in the CL-SL isolate significantly increases parasite survival in the spleen, implicating the homozygous R231C substitution in CL-SL attenuation. finding
- ★ Decreased copy number of the A2 multi-gene family in the CL-SL strain contributes to its attenuation in visceral infection. mechanism
- Comparing genomes of closely related isolates of the same species that cause different human pathologies is a more effective strategy for defining tropism determinants than cross-species comparison. method
- The Sri Lankan CL-SL and VL-SL isolates are more closely related to each other than to the L. donovani reference strain BPK282A1 from Nepal, and both are MON-37 as confirmed by 6PGDH isoenzyme gene sequencing. finding
- Whole-genome and SL-transcriptome datasets for paired cutaneous and visceral L. donovani clinical isolates, including a manually reconstructed A2/A2rel locus that is mis-assembled in the BPK282A1 reference. resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Experimental visceral infection with parasite burden quantification (limiting dilution of spleen homogenates; Leishman Donovan Units for liver) and spleen weight | BALB/c mice (5 mice/group) | Intravenous tail vein injection of 5×10^7 stationary phase promastigotes of CL-SL or VL-SL L. donovani | Spleen weight (splenomegaly), liver parasite burden in LDU, spleen parasite numbers at 4 weeks post-infection | — |
| Experimental cutaneous infection with lesion measurement | BALB/c mice (5 mice/group) | Subcutaneous rear footpad injection of 5×10^6 stationary phase promastigotes of CL-SL or VL-SL | Footpad swelling/lesion development monitored over 11 weeks; parasite numbers | — |
| Transgenic complementation followed by in vivo spleen infection | CL-SL L. donovani promastigotes transfected with VL-SL genes; BALB/c mice | Overexpression/transfection of 7 cloned VL-SL genes (LdBPK171200.1 steroid dehydrogenase, LdBPK220120 phosphoinositide phosphatase, LdBPK252290 DnaJ, LdBPK320370 hypothetical, LdBPK322160 rab-2a, LdBPK341900 NAD-dependent deacetylase, LdBPK366149 Rag C) into CL-SL | Spleen infection level 4 weeks after intravenous infection | — |
| Whole genome sequencing (gDNA-seq) with alignment to reference, CNV/SNP/indel and chromosome somy analysis | CL-SL and VL-SL L. donovani isolates; reference strain BPK282A1 (Nepal) | none | Read coverage in 10 kb tiles (VL/CL ratios), gene copy number, SNPs/indels and their coding effects, chromosome read depth scaled to 2 for disomic chromosomes | Illumina GAIIx, >200× coverage |
| Spliced-leader (SL) cDNA sequencing / transcriptome profiling | L. donovani CL-SL and VL-SL axenic promastigotes, axenic amastigotes, and macrophage-derived amastigotes (infected macrophages) | Life-cycle stage differentiation (promastigote vs axenic amastigote vs intracellular amastigote) | cDNA coverage per chromosome normalized to the entire genome; median chromosome transcript levels | — |
| RT-PCR | Transfected CL-SL L. donovani cells | Transfection with VL-SL genes | Transcription/expression of the corresponding VL-SL transgenes | — |
| Sanger sequencing of isoenzyme gene (6-phosphogluconate dehydrogenase, 6PGDH) | CL-SL and VL-SL L. donovani clinical isolates | none | Sequence-based strain typing confirming both isolates are L. donovani MON-37 | — |
| Sanger sequencing verification of candidate SNPs and manual assembly/read alignment of the A2–A2rel multi-gene family region | CL-SL and VL-SL L. donovani genomic DNA | none | Confirmation of CL-SL-specific non-synonymous SNPs; relative A2 gene copy number between strains | — |
- ▼ VL-SL infection produced splenomegaly and high liver and spleen parasite burdens, whereas CL-SL caused negligible/very little detectable visceral infection
- – In cutaneous infection both isolates showed low virulence, but CL-SL induced transient footpad swelling while VL-SL did not; the swelling was not accompanied by a significant increase in parasite number and was attributed to a stronger inflammatory response
- ▲ Of 7 VL-SL genes transfected into CL-SL, only Rag C significantly increased spleen infection levels 5- to 40-fold across separate experiments
- – The CL-SL Rag C gene carries a homozygous non-conservative R231C substitution at a residue highly conserved across Leishmania and trypanosomatids
- ▲ The A2/A2rel repeat cluster (LdBPK_220670.1, chromosome 22) is present at higher copy number in VL-SL than CL-SL gene VL/CL ratio 1.83
- – Nine regions of copy number variation were identified; higher in VL-SL: LdBPK_111220.1 ABC transporter, LdbpK_161030.1–161110.1 hypothetical cluster, LdbpK_200120.1 phosphoglycerate kinase B, chromosome 27 rRNA locus; higher in CL-SL: chromosome 23 MRPA/YIP1 region, chromosome 1 eIF4a, chromosome 19 glycerol uptake proteins, chromosome 29 hypotheticals tile VL/CL ratios 0.46–2.61; gene ratios 0.35–2.94
- – No gene deletions were detected in either genome, and only 5 genes were unequally affected by frame shift or stop-site change; the sole homozygous CL-SL-specific change (LdBPK_311390.1 stop lost) adds only 3 amino acids
- – Chromosome somy: most chromosomes disomic in both isolates, chromosome 31 tetrasomic and chromosome 23 trisomic in both; chromosomes 13 and 20 trisomic in VL-SL but diploid in CL-SL; chromosome-level transcript levels largely did not track somy (e.g. tetrasomic chromosome 31 had average mRNA levels)
- fold_change 5 to 40 fold increase in spleen infection (CL-SL transfected with VL-SL Rag C vs control, separate experiments, statistically significant)
- fold_change 1.83 (gene VL/CL ratio) (A2 and A2rel repeat cluster copy number, chromosome 22)
- fold_change 2.94 (gene VL/CL ratio); tile ratio 2.61 (LdBPK_111220.1 ABC transporter-like protein, chromosome 11)
- count 117 non-synonymous (70 homozygous + 47 heterozygous) and 92 synonymous (60 homozygous + 32 heterozygous) SNP changes specific to CL-SL; 15 homozygously mutated genes have putative functions (CL-SL-specific coding SNPs vs VL-SL and reference)
- count over 70 pseudogenes from frame shifts or stop codon variations (Common to CL-SL and VL-SL but functional in reference L. donovani BPK282A1)
- other more than 80% of differences common to VL-SL and CL-SL; about 20% of variants heterozygous; about 20% of SNPs in coding regions (Variant comparison against reference strain BPK282A1)
- count 38,243 homozygous and 6,132 heterozygous variants common to VL & CL; 1,977/1,908 unique to VL; 2,251/2,587 unique to CL (Table 2 total SNP/indel counts vs reference genome)
- other >200× coverage; mRNA level for chromosome 9 about 40% higher in VL-SL; 5 mice/group; 5×10^7 promastigotes IV and 5×10^6 subcutaneous (Sequencing depth, chromosome 9 transcript difference, and infection dose/group size)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper compares two Leishmania donovani clinical isolates (CL-SL and VL-SL) primarily through descriptive experimental biology: mouse infection experiments (5 mice/group) reporting organ parasite burdens and footpad swelling as mean plus standard error, a gene-complementation experiment (RagC transfection) described as producing a 'statistically significant' increase in spleen infection without naming the specific test, and genomic/transcriptomic comparisons (SNP/indel counts, copy-number ratios, chromosome somy, RNA-seq coverage) presented largely as descriptive counts and ratios rather than through named inferential statistical tests.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| not specified (result described only as 'statistically significant') | Fig. 2B, spleen infection levels in CL-SL parasites transfected with VL-SL Rag C versus other transfected/control genes | 5 mice per group (as stated for infection experiments); described as replicated in 'separate experiments' | not stated |
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Differences between isolates and transfected lines were summarized as mean plus standard error and described as 'statistically significant' without naming the specific test used or reporting exact p-values.↳ Could also: Explicitly naming the test applied (e.g., Student's t-test or Mann-Whitney U) and reporting exact p-values alongside SEM or a 95% confidence interval. — This would let readers independently judge the strength of evidence and the precision of the estimate, since 'significant' alone does not convey effect magnitude or uncertainty.
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Seven candidate VL-SL genes were transfected individually into CL-SL and each tested for an effect on spleen infection levels.↳ Could also: A multiple-comparison correction such as Bonferroni or Benjamini-Hochberg FDR applied across the panel of tested genes. — Testing several candidate genes within the same experimental round is a setting where a family-wise error rate or false discovery rate correction is commonly used to guard against chance findings among the several comparisons.
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Mouse infection group sizes were small (5 mice/group).↳ Could also: A nonparametric or exact test (e.g., Mann-Whitney U, permutation test) suited to small-sample comparisons. — Such methods do not depend on distributional (e.g., normality) assumptions that can be difficult to verify with small group sizes.
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Chromosome-level transcript abundance was compared between the two isolates (Fig. 3B) using normalized RNA-seq/SL-sequencing coverage, presented descriptively rather than through a formal differential-expression test.↳ Could also: A count-based differential expression framework such as DESeq2 or edgeR with model-based significance testing and FDR control. — These tools model the variance structure of sequencing count data and provide formal per-feature significance and multiple-testing-adjusted estimates across many genes or chromosomes at once.
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Randomization of mice to groups and blinding of outcome assessment (parasite counts, footpad measurements) are not described.↳ Could also: Explicitly stating randomized group allocation and blinded outcome assessment, in line with animal-study reporting frameworks such as ARRIVE. — Documenting these procedures helps readers assess the risk of observer bias in the infection and measurement outcomes.
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SNP/indel and gene copy-number differences between isolates were reported as counts and ratios (Tables 1-3) without a formal enrichment or comparative statistical test.↳ Could also: A statistical enrichment test such as Fisher's exact test to compare the distribution of variant categories (e.g., frame shift, stop gained) between isolates. — This would provide a formal probability estimate of whether the observed differences in variant-class distribution between isolates exceed what would be expected by chance.
What was reproduced
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Assessments & scoring basis
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An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Both datasets (SRP026479 WGS, GSE48475 SL-RNAseq) are fully public and were used 1:1, so this is a genuine reproduction rather than a proxy. Table 3 reproduces exactly (5/5 genotypes and effects) and the two headline CNV loci hold (chr16 1.70-2.13 vs ~1.77-2.00; chr23 0.38-0.42 vs 0.38-0.46), which supports the paper's central tropism thesis. The shortfalls are mostly on our side or in the paper's under-specification: Table 2 counts reach only ~40-60% of reported (Non-Syn unique-VL 85 vs 142) because the original calling/filtering pipeline is never described, and 3/8 Table 4 genes lose signal under our pooled edgeR design instead of the paper's per-stage contrasts. The one substantive unexplained discrepancy is chr19, where the reported 0.49-0.55 depletion is simply not present in the shared reads (0.93-1.03); chr22/A2 is excluded because the paper itself declares it manually curated and non-reproducible. Overall: solid but partial — yellow, not red.
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